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Draft Genome Sequences of Citrobacter freundii Strains CF04 and A41 Isolated from Moribund, Septicemic Giant Gourami (Osphronemus goramy) in Sri Lanka Karim Honein,a S. S. S. De S. Jagoda,b,c Appudurai Arulkanthan,c Hideki Ushio,a Shuichi Asakawab Laboratory of Marine Biochemistry, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japana; Laboratory of Aquatic Molecular Biology and Biotechnology, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japanb; Center for Aquatic Animal Disease Diagnosis and Research, Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lankac K.H. and S.S.S.D.S.J. contributed equally to this work.
Citrobacter freundii is a Gram-negative opportunistic pathogen associated with many infectious conditions including septicemia in humans and animals. Here, we announce the draft genome sequences of two multidrug-resistant C. freundii strains (CF04 and A41) isolated from septicemic giant gourami (Osphronemus goramy) collected from aquaria in Sri Lanka. Received 20 June 2016 Accepted 21 June 2016 Published 11 August 2016 Citation Honein K, Jagoda SSSDS, Arulkanthan A, Ushio H, Asakawa S. 2016. Draft genome sequences of Citrobacter freundii strains CF04 and A41 isolated from moribund, septicemic giant gourami (Osphronemus goramy) in Sri Lanka. Genome Announc 4(4):e00820-16. doi:10.1128/genomeA.00820-16. Copyright © 2016 Honein et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Shuichi Asakawa, [email protected].
C
itrobacter freundii is an infrequent but established cause of systemic infections in farmed food fish and aquarium fish (1, 2). During an investigation to identify septicemia causing bacterial pathogens of fresh water ornamental fish in Sri Lanka, the occurrence of Citrobacter spp. was only second to Aeromonas spp. (3). C. freundii has received increasing attention as an opportunistic pathogen that causes diarrhea, urinary tract infections, and septicemia in humans (4). However, to our knowledge the information on genome sequences of C. freundii isolated from diseased fish was not available. Further, C. freundii has shown a strong ability to developing resistance to antibiotics used in human therapy and aquaculture (5, 6). It is therefore imperative to advance our understanding of pathogenic, multidrug-resistant C. freundii strains associated with fish in order to identify antimicrobial resistance and virulence determinants. Two C. freundii strains, CF04 and A41, were isolated from the kidneys of two moribund giant gourami (Osphronemus goramy) fish showing signs of generalized septicemia collected from aquaria in Kandy, Sri Lanka. Genomic DNA was extracted with the DNeasy Blood and Tissue kit (Qiagen). Genomic DNA libraries were constructed using the Ion Xpress Plus fragment library kit and sequenced on an Ion Torrent PGM (Life Technologies) platform using the ion 318 chip and the 400-bp kit following standard protocols. A total of 1,328,195 reads equivalent to 340,760,015 bp of data and 1,170,722 reads equivalent to 330,995,850 bp of data were, respectively, generated from C. freundii CF04 and A41 DNA samples. The reads acquired were assembled separately using MIRA version 4.0.2 (7) and improved with CLC Genomics Workbench version 8 by alignment against a reference genome (accession no. CP007557). The qualities of the assemblies were evaluated using QUAST version 3.0 (8). The assembly of the C. freundii CF04 strain resulted in 58 con-
July/August 2016 Volume 4 Issue 4 e00820-16
tigs over 1,000 bp (largest contig 1,274,500 bp; N50 ⫽ 679,933 bp) with a total length of 5,138,911 bp and 51.49% G⫹C content. The reads of C. freundii A41 were assembled to 153 contigs larger than 1,000 bp (largest contig 548,564 bp; N50 ⫽ 366,314 bp) with a total length of 5,301,254 bp and 51.5% G⫹C content. The total size of the draft genomes and G⫹C contents are in agreement with published C. freundii genomes (4.9 to 5.0 Mb and 51.6% G⫹C) (9–11). Functional annotation using RAST (12) revealed 5,777 and 5,253 coding sequences (CDS) in the draft genomes of CF04 and A41, respectively. The presence of genes involved with virulence, disease and defense, quorum sensing, and biofilm formation as well as genes encoding resistance to different antibiotics confirms the clinical significance of these isolates. Moreover, RAST indicated that both organisms carry genes encoding resistance to antibiotics that include fluoroquinolon, macrolides, beta-lactams, rifampin, chloramphenicol, tetracycline, aminocoumarin, aminoglycoside, polymyxin, and vancomycin. PHAST, a phage search tool (13), predicted six intact phages in CF04 and seven intact phages in A41. Accession number(s). The draft genome sequences of C. freundii A41 and CF04 have been deposited in DDBJ/EMBL/ GenBank under the accession numbers BDFK01000001 to BDFK01000153 and BDFL01000001 to BDFL01000058, respectively. FUNDING INFORMATION This work, including the efforts of Shuichi Asakawa, was funded by Japan Society for the Promotion of Science (JSPS) (24248034). This work, including the efforts of Shuichi Asakawa, was funded by JST | Core Research for Evolutional Science and Technology (CREST).
REFERENCES 1. Karunasagar I, Karunasagar I, Pai R. 1992. Systemic Citrobacter freundii infection in common carp, Cyprinus carpio L., fingerlings. Fish Sci 15: 95–98. http://dx.doi.org/10.1111/j.1365-2761.1992.tb00642.x.
Genome Announcements
genomea.asm.org 1
Honein et al.
2. Sato N, Yamane N, Kawamura T. 1982. Systemic Citrobacter freundi infection among sunfish Mola mola in Matsushima aquarium. Fish Sci 48:1551–1557. http://dx.doi.org/10.2331/suisan.48.1551. 3. Jagoda S, Honein K, De Silva DPN, Arulkanthan A, Kinoshita S, Ushio H, Asakawa S. 2014. Genetic characterisation of multidrug resistant Citrobacter spp. isolated from septicaemic fresh water ornamental fish, abstr ID279, p. 276. Abstr 9th Symposium on Diseases in Asian Aquaculture, Ho Chi Minh, Vietnam. 4. Schmidt H, Montag M, Bockemühl J, Heesemann J, Karch H. 1993. Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples. Infect Immun 61:534 –543. 5. Kimura K, Kumar S, Takeo M, Mayilraj S. 2014. Genome sequencing, annotation of Citrobacter freundii strain GTC 09479. Genomics Data 2:40 – 41. http://dx.doi.org/10.1016/j.gdata.2013.12.002. 6. Liu X, Huang Y, Xu X, Zhao Y, Sun Q, Zhang Z, Zhang X, Wu Y, Wang J, Zhou D, An X, Pei G, Wang Y, Mi Z, Yin Z, Tong Y. 2016. Complete genome sequence of multidrug-resistant Citrobacter freundii strain P10159, isolated from urine samples from a patient with esophageal carcinoma. Genome Announc 4(1):e01754-15. http://dx.doi.org/10.1128/ genomeA.01754-15. 7. Chevreux B, Wetter T, Suhai S. Genome sequence assembly using trace signals and additional sequence information, p. 45–56. In Computer science and biology. Proceedings of the German Conference on Bioinformatics, GCB ’99. GCB, Hannover, Germany.
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8. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. http://dx.doi.org/10.1093/bioinformatics/btt086. 9. Nawaz M, Khan AA, Khan S, Sung K, Steele R. 2008. Isolation and characterization of tetracycline-resistant Citrobacter spp. from catfish. Food Microbiol 25:85–91. http://dx.doi.org/10.1016/j.fm.2007.07.008. 10. Lü A, Hu X, Zheng L, Zhu A, Cao C, Jiang J. 2011. Isolation and characterization of Citrobacter spp. from the intestine of grass carp Ctenopharyngodon idellus. Aquaculture 313:156 –160. http://dx.doi.org/ 10.1016/j.aquaculture.2011.01.018. 11. Kumar S, Kaur C, Kimura K, Takeo M, Raghava GP, Mayilraj S. 2013. Draft genome sequence of the type species of the genus Citrobacter, Citrobacterfreundii MTCC 1658. Genome Announc 1(1):e00120-12. http:// dx.doi.org/10.1128/genomeA.00120-12. 12. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. http://dx.doi.org/10.1186/ 1471-2164-9-75. 13. Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast phage search tool. Nucleic Acids Res 39:W347–W352. http://dx.doi.org/ 10.1093/nar/gkr485.
Genome Announcements
July/August 2016 Volume 4 Issue 4 e00820-16
Draft Genome Sequences of Citrobacter freundii Strains CF04 and A41 Isolated from Moribund, Septicemic Giant Gourami (Osphronemus goramy) in Sri Lanka Karim Honein,a S. S. S. De S. Jagoda,b,c Appudurai Arulkanthan,c Hideki Ushio,a Shuichi Asakawab Laboratory of Marine Biochemistry, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japana; Laboratory of Aquatic Molecular Biology and Biotechnology, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japanb; Center for Aquatic Animal Disease Diagnosis and Research, Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lankac K.H. and S.S.S.D.S.J. contributed equally to this work.
Citrobacter freundii is a Gram-negative opportunistic pathogen associated with many infectious conditions including septicemia in humans and animals. Here, we announce the draft genome sequences of two multidrug-resistant C. freundii strains (CF04 and A41) isolated from septicemic giant gourami (Osphronemus goramy) collected from aquaria in Sri Lanka. Received 20 June 2016 Accepted 21 June 2016 Published 11 August 2016 Citation Honein K, Jagoda SSSDS, Arulkanthan A, Ushio H, Asakawa S. 2016. Draft genome sequences of Citrobacter freundii strains CF04 and A41 isolated from moribund, septicemic giant gourami (Osphronemus goramy) in Sri Lanka. Genome Announc 4(4):e00820-16. doi:10.1128/genomeA.00820-16. Copyright © 2016 Honein et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Shuichi Asakawa, [email protected].
C
itrobacter freundii is an infrequent but established cause of systemic infections in farmed food fish and aquarium fish (1, 2). During an investigation to identify septicemia causing bacterial pathogens of fresh water ornamental fish in Sri Lanka, the occurrence of Citrobacter spp. was only second to Aeromonas spp. (3). C. freundii has received increasing attention as an opportunistic pathogen that causes diarrhea, urinary tract infections, and septicemia in humans (4). However, to our knowledge the information on genome sequences of C. freundii isolated from diseased fish was not available. Further, C. freundii has shown a strong ability to developing resistance to antibiotics used in human therapy and aquaculture (5, 6). It is therefore imperative to advance our understanding of pathogenic, multidrug-resistant C. freundii strains associated with fish in order to identify antimicrobial resistance and virulence determinants. Two C. freundii strains, CF04 and A41, were isolated from the kidneys of two moribund giant gourami (Osphronemus goramy) fish showing signs of generalized septicemia collected from aquaria in Kandy, Sri Lanka. Genomic DNA was extracted with the DNeasy Blood and Tissue kit (Qiagen). Genomic DNA libraries were constructed using the Ion Xpress Plus fragment library kit and sequenced on an Ion Torrent PGM (Life Technologies) platform using the ion 318 chip and the 400-bp kit following standard protocols. A total of 1,328,195 reads equivalent to 340,760,015 bp of data and 1,170,722 reads equivalent to 330,995,850 bp of data were, respectively, generated from C. freundii CF04 and A41 DNA samples. The reads acquired were assembled separately using MIRA version 4.0.2 (7) and improved with CLC Genomics Workbench version 8 by alignment against a reference genome (accession no. CP007557). The qualities of the assemblies were evaluated using QUAST version 3.0 (8). The assembly of the C. freundii CF04 strain resulted in 58 con-
July/August 2016 Volume 4 Issue 4 e00820-16
tigs over 1,000 bp (largest contig 1,274,500 bp; N50 ⫽ 679,933 bp) with a total length of 5,138,911 bp and 51.49% G⫹C content. The reads of C. freundii A41 were assembled to 153 contigs larger than 1,000 bp (largest contig 548,564 bp; N50 ⫽ 366,314 bp) with a total length of 5,301,254 bp and 51.5% G⫹C content. The total size of the draft genomes and G⫹C contents are in agreement with published C. freundii genomes (4.9 to 5.0 Mb and 51.6% G⫹C) (9–11). Functional annotation using RAST (12) revealed 5,777 and 5,253 coding sequences (CDS) in the draft genomes of CF04 and A41, respectively. The presence of genes involved with virulence, disease and defense, quorum sensing, and biofilm formation as well as genes encoding resistance to different antibiotics confirms the clinical significance of these isolates. Moreover, RAST indicated that both organisms carry genes encoding resistance to antibiotics that include fluoroquinolon, macrolides, beta-lactams, rifampin, chloramphenicol, tetracycline, aminocoumarin, aminoglycoside, polymyxin, and vancomycin. PHAST, a phage search tool (13), predicted six intact phages in CF04 and seven intact phages in A41. Accession number(s). The draft genome sequences of C. freundii A41 and CF04 have been deposited in DDBJ/EMBL/ GenBank under the accession numbers BDFK01000001 to BDFK01000153 and BDFL01000001 to BDFL01000058, respectively. FUNDING INFORMATION This work, including the efforts of Shuichi Asakawa, was funded by Japan Society for the Promotion of Science (JSPS) (24248034). This work, including the efforts of Shuichi Asakawa, was funded by JST | Core Research for Evolutional Science and Technology (CREST).
REFERENCES 1. Karunasagar I, Karunasagar I, Pai R. 1992. Systemic Citrobacter freundii infection in common carp, Cyprinus carpio L., fingerlings. Fish Sci 15: 95–98. http://dx.doi.org/10.1111/j.1365-2761.1992.tb00642.x.
Genome Announcements
genomea.asm.org 1
Honein et al.
2. Sato N, Yamane N, Kawamura T. 1982. Systemic Citrobacter freundi infection among sunfish Mola mola in Matsushima aquarium. Fish Sci 48:1551–1557. http://dx.doi.org/10.2331/suisan.48.1551. 3. Jagoda S, Honein K, De Silva DPN, Arulkanthan A, Kinoshita S, Ushio H, Asakawa S. 2014. Genetic characterisation of multidrug resistant Citrobacter spp. isolated from septicaemic fresh water ornamental fish, abstr ID279, p. 276. Abstr 9th Symposium on Diseases in Asian Aquaculture, Ho Chi Minh, Vietnam. 4. Schmidt H, Montag M, Bockemühl J, Heesemann J, Karch H. 1993. Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples. Infect Immun 61:534 –543. 5. Kimura K, Kumar S, Takeo M, Mayilraj S. 2014. Genome sequencing, annotation of Citrobacter freundii strain GTC 09479. Genomics Data 2:40 – 41. http://dx.doi.org/10.1016/j.gdata.2013.12.002. 6. Liu X, Huang Y, Xu X, Zhao Y, Sun Q, Zhang Z, Zhang X, Wu Y, Wang J, Zhou D, An X, Pei G, Wang Y, Mi Z, Yin Z, Tong Y. 2016. Complete genome sequence of multidrug-resistant Citrobacter freundii strain P10159, isolated from urine samples from a patient with esophageal carcinoma. Genome Announc 4(1):e01754-15. http://dx.doi.org/10.1128/ genomeA.01754-15. 7. Chevreux B, Wetter T, Suhai S. Genome sequence assembly using trace signals and additional sequence information, p. 45–56. In Computer science and biology. Proceedings of the German Conference on Bioinformatics, GCB ’99. GCB, Hannover, Germany.
2 genomea.asm.org
8. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. http://dx.doi.org/10.1093/bioinformatics/btt086. 9. Nawaz M, Khan AA, Khan S, Sung K, Steele R. 2008. Isolation and characterization of tetracycline-resistant Citrobacter spp. from catfish. Food Microbiol 25:85–91. http://dx.doi.org/10.1016/j.fm.2007.07.008. 10. Lü A, Hu X, Zheng L, Zhu A, Cao C, Jiang J. 2011. Isolation and characterization of Citrobacter spp. from the intestine of grass carp Ctenopharyngodon idellus. Aquaculture 313:156 –160. http://dx.doi.org/ 10.1016/j.aquaculture.2011.01.018. 11. Kumar S, Kaur C, Kimura K, Takeo M, Raghava GP, Mayilraj S. 2013. Draft genome sequence of the type species of the genus Citrobacter, Citrobacterfreundii MTCC 1658. Genome Announc 1(1):e00120-12. http:// dx.doi.org/10.1128/genomeA.00120-12. 12. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. http://dx.doi.org/10.1186/ 1471-2164-9-75. 13. Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast phage search tool. Nucleic Acids Res 39:W347–W352. http://dx.doi.org/ 10.1093/nar/gkr485.
Genome Announcements
July/August 2016 Volume 4 Issue 4 e00820-16
